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M. Levrero - Histone Acetyltransferase (HAT) autoregulatory loops in the regulation of DNA damage responses<br />
modulation of chromatin-bound hSirT1 deacetylase<br />
activity by the intracellular redox state with P1p73<br />
promoter activity. Release of PCAF from hSirT1<br />
repression favours the assembly of transcriptionally<br />
active PCAF/E2F1 complexes onto the P1p73 promoter<br />
and p53-independent apoptosis. These results<br />
identify hSirT1 and PCAF as potential targets to<br />
modulate tumor cell survival and chemoresistance<br />
irrespective to p53 status.<br />
Chromatin Immunoprecipitation-based<br />
innovative reagents for a comprehensive<br />
evaluation of the E2F1 transcriptome<br />
Microarrays-based expression profiling studies have<br />
led to the identification of hundreds potential direct<br />
target genes activated and/or repressed by the different<br />
members of the E2F family of transcription<br />
factors. Genome-wide ChIP-chip experiments have<br />
confirmed that several hundreds putative E2F binding<br />
sites exist and are potentially bound throughout<br />
the genome. We combined an extensive in silico<br />
analysis with literature survey to select 327 putative<br />
E2F1 direct target genes containing highly conserved<br />
E2F/DP binding sites in their promoter or<br />
first intron (i.e. -2500 to +1000 respect to tss). To<br />
build up the E2F/DP dedicated microchip slide 3 different<br />
45-50mer oligos for each of the 327 target<br />
gene were designed. Each oligo was spotted in triplicate<br />
and the resulting array printed 4 times on the<br />
same slide for a total of 11772 spots. Gene ontology<br />
analysis of the 327 selected genes is in agreement<br />
with the notion that the spectrum of direct E2Fs target<br />
genes might be far more variated than previously<br />
thought: apoptosis (31), DNA damage (30), cell<br />
cycle (71) cell growth (16), signal transduction (29);<br />
transcription regulation (73), ubiquitination/Protein<br />
degradation (7), protein/cell metabolism (30);<br />
cytoskeleton/cell adhesion (38).<br />
The E2F-ChIP array has been validated by<br />
hybridization with anti-E2F1 and anti-Ac-H3<br />
immunoprecipitated chromatin derived from untreat-<br />
58<br />
ed and doxorubicin treated U2OS cells. The analysis<br />
of 2 independent ChIP-chip experiments has<br />
revealed that E2F1 is present on 48% of the genes in<br />
untreated cells. In cells exposed to apoptotic dosages<br />
of doxorubicin, E2F1 is released from 33 out of 157<br />
genes that were occupied in untreated genes (10% out<br />
of 48%) whereas E2F is recruited onto 118 additional<br />
target genes (36%) that were not bound in untreated<br />
cells. There results indicate that DNA damage<br />
results in a wide change in the spectrum of E2F1<br />
bound promoters. E2F1 is bound before and after<br />
treatment on 41% of the genes involved in apoptosis<br />
and DNA repair included in the array and it is<br />
recruited on additional 38% of these genes.<br />
Interestingly, among the genes that regulate cell<br />
cycle and whose expression is expected to be mostly<br />
down regulated in cells undergoing DNA damageinduced<br />
growth-arrest and apoptosis, only 17% loose<br />
E2F1 after exposure to doxorubicin whereas E2F1<br />
remains bound to 30% of these genes and is recruited<br />
on 39% additional genes. The analysis of the correlation<br />
between E2F1 promoter occupancy and gene<br />
expression indicate that multiple mechanisms are<br />
involved in the regulation of E2F target genes after<br />
DNA damage including the recruitment of repressive<br />
E2F1-containing complexes and the modulation<br />
of E2F1-bound complexes by the recruitment of corepressors<br />
and the release of coactivators. The E2F<br />
ChIP-on-chip reagent we have developed and validated<br />
and the knowledge that it generates will represent<br />
a powerful tool to define the spectrum of genes targeted<br />
by E2F1 to modulate cell death, senescence and<br />
proliferation and to functionally characterize the<br />
E2F transcriptome in vitro and in vivo.<br />
Selected publications<br />
Blandino G, Fanciulli M, Levrero M, Piaggio G.<br />
The post-genomic era: workshop on chromatin<br />
immunoprecipitation-related techniques. Cell Death<br />
Differ. 2007, 14:1390-1.